顺铂通过调控P53及P21表达水平影响HepG2细胞衰老及周期阻滞(英文)

Cisplatin induces cell cycle arrest and senescence via upregulating P53 and P21 expression in HepG2 cells

目的化疗药物能够通过诱导肿瘤细胞衰老从而发挥治疗效果。顺铂作为最常用的化疗药物能否诱导肝癌细胞发生加速性衰老目前尚不清楚。方法使用MTT法和克隆形成试验检测不同剂量顺铂对HepG2细胞增殖的影响;分别用流式细胞仪及衰老相关β-半乳糖苷酶染色检测细胞周期及衰老情况;RT-PCR检测TP53、P21及P19基因的mRNA表达水平,Western blotting检测P53和P21蛋白表达水平。结果顺铂能够诱导HepG2细胞出现不可逆的生长停滞及细胞周期阻滞。2.0μg/ml顺铂作用于HepG2后衰老相关β-半乳糖苷酶染色阳性,并呈现时间依赖性。在顺铂诱导衰老过程中,P19基因表达水平升高,在诱导48 h后达到峰值后逐渐下降,而P53和P21表达水平则持续升高。结论本研究提示顺铂能够诱导肝癌细胞出现衰老样表型,这一结果为进一步探讨其抗肝癌机制提供了基础。

Objective Cellular senescence as one of the important steps against tumor is observed in many cancer patients receiving chemotherapy and is related to chemotherapeutic response. To investigate the effect of cisplatin on hepatocellular carcinoma, we treated HepG2 cells exhibiting wild-type TP53 with gradient concentrations of cisplatin. Methods The inhibitory effects of cisplatin on human hepatoma HepG2 cells were detected by MTT assay and colony formation test. The changes in cell cycle were analyzed by flow cytometry, and cellular senescence was detected with senescence associatedβ-galactosidase （SA β-gal） staining. The relative mRNA expression levels of TP53, P21 and P19 was estimated using semi-quantitative real-time RT-PCR, and the protein expressions of P53 and P21 were detected using Western blotting. Results Cisplatin induced irreversible proliferation inhibition and G1 phase arrest of HepG2 cells. Elevated levels of senescence-associated β-galactosidase was observed in HepG2 cells exposed to low doses of cisplatin. P19 expression immediately increased following cisplatin exposure and reached the maximum level at 48 h, followed then by a rapid decrease to the baseline level, whereas the expressions levels of TP53 and P21 mRNA increased continuously. Western blotting confirmed P53 and P21 expression changes similar to their mRNA expressions during cisplatin-induced cellular senescence in HepG2 cells. Conclusion Our results revealed a functional link between cisplatin and hepatocellular senescence. Cellular senescence induced by cisplatin as a stabile senescent cellular model can be used for further research.